Affinity tag nucleic acid and protein compositions, and processes for using same

ABSTRACT

The present invention concerns compositions and processes that use affinity tags for isolating, and detecting or quantifying analytes, including nucleic acids, proteins and polypeptides. Compositions include nucleic acid compositions and protein compositions with affinity binding pairs, including metal binding peptides and immobilized metals, or peptide affinity groups.

FIELD OF THE INVENTION

This invention relates to affinity tag compositions including affinitytag nucleic acids and proteins, and processes useful for isolating anddetecting or quantifying species of a nucleic acid of interest, andother processes for modifying, isolating, detecting or quantifyingproteins and analytes of interest.

All patents, patent applications, patent publications, scientificarticles and the like, cited or identified in this application arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the present invention pertains.

BACKGROUND OF THE INVENTION

For many purposes of manipulating or analyzing nucleic acids, the firstimportant step is isolation of the nucleic acids from other cellularmaterial. In this regard, the earliest methods were relatively crudemethods using ethanol precipitation followed by phase partitioning withorganic reagents. For instance, phenol has been widely used to separateDNA from cellular material while RNA is more commonly isolated using aguanidinium isothiocyanate/phenol/chloroform mixture. These methods donot depend on the particular sequences of the nucleic acids for theirisolation, i.e., they are sequence independent and the basis ofseparation is strictly derived from general chemical properties of DNAand RNA.

More sophisticated methods were later developed that employed theparticular sequences of the nucleic acids as an identifying feature forseparation, thereby enabling the isolation of nucleic acids withselected sequences apart from other nucleic acids as well as from othercellular material. A notable example of this method is “hybrid capture”where a nucleic acid complementary to the sequence or sequences ofinterest is used to specifically hybridize to one or more target nucleicacids. At a later step, a tag on the capture probe is used to separatematerial that has hybridized to the capture probe from material thatremained unhybridized. Examples of formats that exploit this methodologyinclude beads with oligo T segments for isolation of polyA RNA, andstrepavidin-coated microtitre plates that can bind biotinylated primersafter amplification reactions. In either case, a moiety capable ofbinding the tag is fixed to a solid support, thus enabling a series ofsimple washing steps to remove nucleic acids lacking the sequences ofinterest. Thus, in one case, a nucleic acid sequence is added to thecapture probe, and in the other case, one of the nucleotides is modifiedby the addition of a ligand. Unfortunately, these methods aredisadvantaged by the slower kinetics of mixed phase hybridization in thefirst case and the low capacity engendered by the attachment of largebulky proteins to a solid matrix in the aforementionedbiotin/strepavidin method.

While conceptually simple, the isolation and purification of proteinshas been at the same time both easier and more problematic. Unlikenucleic acids that have similar chemical properties regardless ofsequence differences, the variety of different amino acids and theexistence of secondary and tertiary structures have allowed theapplication of various criteria to be used for isolation of a singlespecies of protein. These criteria include differences in molecularweight, shape, salt solubility, net charge and polar versus nonpolarcharacteristics. Thus, for purification of any given protein, a seriesof separation steps can be carried out that will be unique to thatparticular protein. However, these standard methods of proteinpurification lack the advantages described earlier for isolation ofunique nucleic acid sequences where essentially a single methodology canbe applied to purification of any species of interest. Although this hasremained true for most native proteins, the burgeoning field ofrecombinant DNA has allowed more flexibility in modifying desirableproteins such that they carry additional amino acid sequences that canbe helpful during purification procedures. The most notable example ofsuch methods is the histidine tag which has been added to either thecarboxy or amino end of the coding sequence (Dobeli et al., U.S. Pat.No. 5,284,933). The important feature of this oligopeptide sequence isthat it has an affinity for chelated metals, such that a matrix withimmobilized metal can be used to bind any protein that has such ahistidine tag (Dobeli et al., U.S. Pat. No. 4,877,830), a methodcommonly referred to as IMAC (Immobilized Metal AffinityChromatography). Thus, a single isolation procedure can be used for awide variety of proteins after the proteins have been suitably modified.Although oligohistidine is the best known example of an oligo peptidethat can bind to an immobilized metal, other petides have been describedas well, including one that has the amino acid sequence HGGHHG (Cheng etal. 2004 Bio-organic & Medicinal Chemistry Letters 14; 1987-1990)

The use of non-nucleic acid affinity tags has also been used inconjunction with nucleic acids. For instance, Min and Verdine (1996Nucleic Acids Research 24:3806-3810) have described a nucleic acidprimer with modified bases at the 5′ end with histidine moietiesattached to the bases. As such, their primer does not contain anoligopeptide tag as described above, but rather the 5′ end has beenmodified with a series of histaminyl purine residues. Extension of theseprimers in a PCR reaction allows collection of the PCR products by meansof a chelated resin. No application is described in this publication,however, for using these constructs for either signal detection oranalyte isolation.

Stanley et al. (U.S. Pat. No. 5,843,663) describe the use of affinityagents attached to peptide nucleic acids (PNAs). As described previouslyin Min and Verdine (1996), cited supra., the individual amino acids areattached to each nucleotide analog as opposed to a true oligohistidinecapture agent. It also should be pointed out that this is not an exampleof a chimeric molecule consisting of a nucleic acid and an affinity tagbecause the peptide nucleic acid is actually a synthetic substitute fora nucleic acid. The backbones of the constructs described by Stanley etal. have an essentially homogeneous nature because both the subunits ofthe amino acid segment and the pleptide nucleic acid analogue segmentare joined together by a succession of peptide bonds to form a singlepolymeric molecule. The method described in this patent has drawbacksthat are intrinsic to the use of peptide nucleic acids. Specifically,efficient synthesis is limited to only short PNA sequences and there isa high cost associated with the reagents used in PNA synthesis.

Soderlund et al. (U.S. Patent Appl. No. 20040053300) describe a methodof determining the quantity of discrete polynucleotide analytes by theuse of a pool of nucleic acid probes of various sizes. The probeshybridize to analytes that have been modified by the addition of anaffinity tag (such as oligo histidine) to the base portion. Afterhybridization of the probes to analytes, complexes are isolated byvirtue of the presence of the affinity agent in the analyte allowingbinding to a matrix. In a subsequent step the bound probes are releasedand quantified, thus giving an indirect measurement of the amount ofanalytes present in a sample. In this particular instance, the analytesthemselves have been covalently attached to an affinity agent.

Affinity binding pairs have also been used in conjunction with RNAmolecules in Krause and Simmons (U.S. Patent Appl. No. 20060105341). Inthis application, the use of a so-called RNA “fusion” molecule with “RNAtags” is described. In this particular case, however, the “fusion” isnot RNA linked to a non-nucleic acid but rather the molecule is a fusionof different nucleic acid sequences resulting in a homogenous nucleicacid where a first RNA segment with a protein binding sequences isappended to a second RNA segment with a selected nucleic acid sequence.This second RNA segment may bind, in turn, to a fusion protein with twodomains where one domain binds the RNA tag and the other domain can bean affinity partner, such as an oligo-His tag, that can be used to bindthe RNA protein complex to a matrix. This composition has been used foridentification and purification of RNA protein complexes and it has notbeen used for signal generation or isolation of nucleic acid analytes.

Histidine has also been used for other purposes besides an affinitylabel. For example, Van Ness et al. (U.S. Pat. No. 7,247,434) describemethods for simultaneously determining a number of different nucleicacid sequences by the use of tagged nucleic acid fragments. Sequencesare derived from the association of a different tag for each nucleotidebase incorporated into nucleic acids synthesized from analyte templates.In one particular instance, a single histidine moiety is used as one ofthe base-specific tags where identification is carried out by massspectrometry after the nucleic acids have been separated by length. Inthis particular instance the histidine is not being used as an affinityagent but only as an identifier tag.

Many of the drawback in the previous uses of affinity tags such ashistidine tags are overcome by the present invention.

SUMMARY OF THE INVENTION

This invention provides a composition which comprises a nucleic acid andone member of an affinity binding pair, wherein the member is attachedto one or more nucleotides of the nucleic acid through a phosphate orsugar of the nucleotide or nucleotides.

This invention also provides a composition just described wherein theaffinity binding pair comprises: (a) a metal binding peptide and animmobilized metal, or (b) a peptide affinity group.

This invention additionally provides a chimeric nucleic acid comprisingat least two portions, a first portion comprising a nucleic acidcomplementary to a nucleic acid sequence of interest, and a secondportion comprising a metal binding peptide, wherein the metal bindingpeptide is attached to one or more nucleotides of the nucleic acid inthe first portion through a sugar or phosphate of the nucleotide ornucleotides.

Also provided by this invention is a chimeric nucleic acid comprising atleast two portions, a first portion comprising a nucleic acidcomplementary to a nucleic acid sequence of interest, and a secondportion comprising one member of a peptide affinity group, wherein themember is attached to one or more nucleotides of the nucleic acid in thefirst portion.

The present invention provides a process for isolating one or morespecies of a nucleic acid of interest. Various steps are used includingthe first step of providing a sample containing or suspected ofcontaining the nucleic acid of interest, a composition which comprises anucleic acid portion and a first member of an affinity binding pair,wherein the nucleic acid portion comprises sequences complementary tothe nucleic acid species of interest, wherein the affinity binding paircomprises (a) a metal binding peptide and an immobilized metal, or (b) apeptide affinity group; and wherein the first member of the affinitybinding pair is attached to one or more nucleotides in the nucleic acidportion; and a matrix comprising a second member of the affinity bindingpair. The composition hybridizes with any nucleic acid of interestcontained in the sample to form a first complex. The first complex iscontacted with the matrix to form a second complex by means of a bindinginteraction between the first member and the second member of theaffinity binding pair. Bound material is separated from unboundmaterial, thereby isolating the nucleic acid species of interest.

The present invention also provides a process for detecting the presenceor quantity of a nucleic acid of interest. In an initial step, thefollowing elements are provided: a sample containing labeled nucleicacids, a composition comprising a nucleic acid portion and a firstmember of an affinity binding pair, wherein the nucleic acid portioncomprises sequences complementary to the nucleic acid of interest, and amatrix comprising a second member of the affinity binding pair; whereinthe affinity binding pair comprises (a) a metal binding peptide and animmobilized metal, or (b) a peptide affinity group; and wherein thefirst member of the affinity binding pair is attached to one or morenucleotides of the nucleic acid portion. The composition is allowed tohybridize with any nucleic acid of interest contained in the sample toform a first complex. This first complex is contacted with the matrix toform a second complex by means of a binding interaction between thefirst member and the second member of the affinity binding pair. Thematrix is washed to remove unhybridized nucleic acids from the matrix.Detecting or quantifying the nucleic acid of interest is carried out bymeans of detecting or quantifying a signal from the labels.

The present invention provides yet another process for detecting thepresence or quantity of a nucleic acid of interest. Various steps areperformed including the initial step of providing the followingelements: a sample containing or suspected of containing the nucleicacid of interest; a labeled probe complementary to the nucleic acid ofinterest; a composition comprising a nucleic acid portion and a firstmember of an affinity binding pair, wherein the nucleic acid portioncomprises sequences complementary to the nucleic acid of interest,wherein the affinity binding pair comprises (a) a metal binding peptideand an immobilized metal, or (b) a peptide affinity group, and whereinthe first member of the affinity binding pair is attached to one or morenucleotides of the nucleic acid portion through a sugar, phosphate orbase of the nucleotide or nucleotides; and a matrix comprising a secondmember of the affinity binding pair. Any nucleic acids of interest inthe sample are allowed to hybridize with labeled probe and thecomposition to form a first complex. This first complex is contactedwith the matrix to form a second complex by means of a bindinginteraction between the first member and the second member of theaffinity binding pair. The matrix is washed to remove unbound materialsfrom the sample. The nucleic acid of interest is detected or quantifiedby means of detecting or quantifying a signal from the labels.

Yet another process provided by the present invention is one fordetecting the presence or quantity of a nucleic acid of interest.Various steps are performed including the initial step of providing asample containing or suspected of containing nucleic acid of interest; aprobe complementary to the nucleic acid of interest and comprising twoportions, wherein a first comprises sequences complementary to thenucleic acid of interest, and a second portion comprising a signalsequence; a composition comprising a nucleic acid portion and a firstmember of an affinity binding pair, wherein the nucleic acid portioncomprises sequences complementary to the nucleic acid of interest,wherein the affinity binding pair comprises (a) a metal binding peptideand an immobilized metal, or (b) a peptide affinity group, and whereinthe first member of the affinity binding pair is attached to one or morenucleotides of the nucleic acid; and a matrix comprising a second memberof the affinity binding pair. Any nucleic acids of interest in thesample are hybridized with labeled probe and the composition to form afirst complex. The first complex is contacted with the matrix to form asecond complex by means of a binding interaction between the one or morebinding partners and the affinity peptide. The matrix is washed toremove unbound materials from the sample. The nucleic acid of interestis detected or quantified by hybridizing labeled oligonucleotidescomplementary to the signal sequence.

The present invention also provides a fusion protein comprising abiologically active polypeptide or protein and at least one affinitypeptide attached to the amino-terminus or the carboxyl-terminus of thebiologically active polypeptide or protein, wherein the affinity peptidecomprises at least a portion of the amino acid sequence of kininogen,such portion comprising a metal binding peptide.

The present invention additionally provides a fusion protein comprisinga biologically active polypeptide or protein and an affinity peptideattached at the amino-terminus and an affinity peptide attached at thecarboxyl-terminus of the biologically active polypeptide or protein,wherein the affinity peptides comprise at least a portion of the aminoacid sequence kininogen, such portion comprising a metal bindingpeptide.

Another composition provided by this invention is a fusion proteincomprising an antibody linked by its amino- and/or carboxyl-terminus toone or two affinity peptides, wherein the affinity peptide binds to ametal, and wherein the antibody has an affinity to an epitope on adifferent antibody.

The invention herein provides a process for modifying a protein ofinterest, this process comprising the steps of first providing (i) anucleic acid that codes for the protein of interest; (ii) a nucleic acidthat codes for a portion of the amino acid sequence of kininogen,wherein that portion codes for a metal binding peptide; and (iii) anexpression vector. The nucleic acid (ii) is added to said nucleic acid(i) to generate a nucleic acid coding for a fusion protein. The nucleicacid coding for the fusion protein is inserted into the expressionvector (iii), thereby generating a vector that expresses the modifiedprotein of interest.

Additionally the invention herein provides a process for isolating aprotein of interest, and this process comprises an initial step ofproviding: (i) a nucleic acid that codes for the protein of interest;(ii) a nucleic acid that codes for a portion of the amino acid sequenceof kininogen, wherein the portion codes for a metal binding peptide;(iii) an expression vector; and (iv) a metal-modified matrix. Othersteps include adding the nucleic acid (ii) to the nucleic acid (i) togenerate a nucleic acid coding for a fusion protein, and inserting thenucleic acid coding for the fusion protein into the expression vector(iii), thereby generating a vector that expresses the protein ofinterest. Finally, the modified protein of interest is purified bybinding the protein of interest to the metal-modified matrix (iv).

Other compositions are provided by the present invention including afusion protein comprising an antibody and at least one affinity peptideattached to the amino terminus or the carboxyl terminus of the antibody,wherein the affinity peptide comprises a metal binding peptide or is onemember of a peptide affinity group, wherein the antibody has an affinityfor a different antibody.

Yet another fusion protein provided by this invention is one comprisingan antibody and an affinity peptide attached to the amino terminus andan affinity peptide attached to carboxyl terminus of the antibody,wherein the affinity peptide comprises a metal binding peptide or is onemember of a peptide affinity group, wherein the antibody has an affinityfor a different antibody.

The invention herein also provides a process for isolating an analyte ofinterest, the process comprising the initial step of providing (i) asample containing or suspected of containing the analyte of interest;(ii) a first antibody having an affinity for the analyte; (iii) a fusionantibody comprising: (a) an antibody and at least one affinity peptideattached to the amino terminus or the carboxyl terminus of the antibody,wherein the affinity peptide comprises a metal binding peptide or is onemember of a peptide affinity group, wherein the antibody has an affinityfor a different antibody; or (b) an antibody and an affinity peptideattached to the amino terminus and an affinity peptide attached tocarboxyl terminus of the antibody, wherein the affinity peptidecomprises a metal binding peptide or is one member of a peptide affinitygroup, wherein the antibody has an affinity for a different antibody;and (iv) a matrix comprising a metal or a second member of the affinitypeptide group. The sample is contacted with the first antibody, therebyforming a first complex between the first antibody and any analytepresent in the sample. The first complex is complexed with the fusionantibody, thereby forming a second complex between the first complex andthe fusion antibody. The second complex is contacted with the matrix tobind the second complex to the matrix. Unbound material is removed fromthe matrix. The analyte of interest is released from the second complex,thereby isolating the analyte of interest.

Another process provided by this invention is one for detecting orquantifying an analyte of interest, said process comprising varioussteps. The first step provides (i) a labeled sample containing orsuspected of containing labeled analyte of interest; (ii) a firstantibody having an affinity for the analyte; (iii) a fusion antibodycomprising: (a) an antibody and at least one affinity peptide attachedto the amino terminus or the carboxyl terminus of the antibody, whereinthe affinity peptide comprises a metal binding peptide or is one memberof a peptide affinity group, wherein the antibody has an affinity for adifferent antibody; or (b) an antibody and an affinity peptide attachedto the amino terminus and an affinity peptide attached to carboxylterminus of the antibody, wherein the affinity peptide comprises a metalbinding peptide or is one member of a peptide affinity group, whereinthe antibody has an affinity for a different antibody; and (iv) a matrixcomprising a metal or a second member of the affinity peptide group. Thesample is contacted with the first antibody, thereby forming a firstcomplex between the first antibody and any analyte present in thesample. The first complex is contacted with the fusion antibody, therebyforming a second complex between the first complex and the fusionantibody. The second complex is contacted with the matrix to bind thesecond complex to the matrix. Unbound material is removed from thematrix. Labeled analytes bound to the matrix are detected or quantifiedby means of detecting or quantifying a signal from the labels.

Yet another process provided herein is one for detecting or quantifyingan analyte of interest, the process comprising various steps includingthe first step of providing (i) a labeled sample containing or suspectedof containing labeled analytes of interest; (ii) a first antibody havingan affinity for the analyte; (iii) a fusion antibody comprising: (a) anantibody and at least one affinity peptide attached to the aminoterminus or the carboxyl terminus of the antibody, wherein the affinitypeptide comprises a metal binding peptide or is one member of a peptideaffinity group, wherein the antibody has an affinity for a differentantibody; or (b) an antibody and an affinity peptide attached to theamino terminus and an affinity peptide attached to carboxyl terminus ofthe antibody, wherein the affinity peptide comprises a metal bindingpeptide or is one member of a peptide affinity group, wherein theantibody has an affinity for a different antibody; and (iv) a matrixcomprising a metal or a second member of the affinity peptide group. Afirst complex is formed among the matrix, the fusion antibody and thefirst antibody. The first complex is contacted with the labeled sample,thereby forming a second complex between the first complex and anylabeled analytes that may be present in the sample. Unbound material isremoved from the matrix. The labeled analytes bound to the matrix aredetected or quantified by means of detecting or quantifying a signalfrom the labels.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts various format that could be used with chimeric primers.

FIG. 1A illustrates a chimeric construct with a first portion consistingof a nucleic acid complementary to a chosen nucleotide sequence and asecond portion with an oligohistidine portion used to bind nucleic acidswith the chosen sequences to a solid matrix thereby allowing isolationof either nucleic acids that bind to the matrix or nucleic acids thatlack complementarity to the construct.

FIG. 1B depicts a format where a chimeric construct similar to the onein FIG. 1A is used to detect the presence of the complementary sequencewhen a collection of labeled analytes are allowed to hybridize to theconstruct.

FIG. 1C shows a chimeric construct similar to the one in FIG. 1A that isused to detect the presence of unlabeled analytes by by means of a probecomplementary to the sequence of interest.

FIG. 1D illustrates a chimeric construct having energy transfer elementswhere hybridization of an analyte labeled with energy transfer elementsprovides signal generation that is dependent upon hybridization of theanalyte to the construct.

FIG. 1E is a depiction of a format where energy transfer takes placebetween a labeled analyte and a signal probe.

FIG. 1F is a depiction of a format where the analyte is unlabled andanalyte specific energy transfer takes place between a signal probe anda chimeric construct.

FIG. 2 is illustrative of binding of chimeric constructs to matrices.

FIG. 2A shows binding and elution of labeled chimeric constructs with Nicolumn.

FIG. 2B shows binding of labeled chimeric constructs with a 96 wellplate.

FIG. 3 illustrates the effects of various reagents on binding ofchimeric constructs.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a composition which comprises a nucleic acidportion that provides specific hybridization to a nucleic acid analyteof interest and a non-nucleic acid portion that comprises at least onemember of an affinity binding pair that allows capture of thecomposition to a solid matrix wherein the member is attached to one ormore nucleotides of the nucleic acid and this attachment can be throughthe phosphate, sugar or base of the nucleotide or nucleotides. Amongsuch affinity binding pairs contemplated by this invention are pairscomprising an immobilized metal and a peptide or oligopeptde that has anaffinity for such a metal.

Thus, the present invention provides a composition which comprises anucleic acid and one member of an affinity binding pair, wherein themember is attached to one or more nucleotides of the nucleic acidthrough a phosphate or sugar of the nucleotide or nucleotide, and suchattachment to such nucleotide or nucleotides can be through a linker armas described further below. Furthermore, in the present composition andinvention, the affinity binding pair comprises: (a) a metal bindingpeptide and an immobilized metal, or (b) a peptide affinity group. In apreferred embodiment the metal is immobilized by chelation. In thepresent invention, a peptide or oligopeptide is defined as a successionof amino acids joined through peptide bonds. Examples of metals that maybe bound by such peptides are nickel, copper, cobalt and zinc. Examplesof such peptides are oligohistidine and an oligopeptide with thesequence HGGHHG that have been referred to earlier. Other sucholigopeptides that may be of use can include SPHHG, SPHHGGSPHHG, HPHHG,HPHHGGHPHHG, SPHHGGHPHHG and HPHHGGSPHHG described by Pasquinelli etal., 2000 (Biotechnol. Prog. 16, 86-91), KDHLIHNVHKEEHAHAHNK describedby Chaga et al., 1999 (J. Chromatog A. 864; 247-256) as well assequences derived from domain 5 of kininogen such as HGLGHGHEQQHGLGHGHand GHGLGHGHEQQHGLGHGHK (DeLa Cadena et al., 1992 Protein Science 1;151-160; Pixley et al., 2003 J Thrombosis and Haemostasis 1; 1791-1798;and Herwald et al., 2001 Eur J Biochem 268; 396-404), all of which areincorporated by reference. Thus, the metal binding peptide can compriseany of the aforementioned amino acid sequences: oligohistidine, HGGHHG,SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG, SPHHGGHPHHG, HPHHGGSPHHG,KDHLIHNVHKEEHAHAHNK, GHGLGHGHEQQHGLGHGHK or HGLGHGHEQQHGLGHGH.

Other affinity binding pairs that may find use with the presentinvention can include peptide affinity pairs. In the present invention apeptide affinity pair is defined as any binary combination of peptides,oligopeptides or proteins that that are capable of recognizing andbinding to each other. Examples of such pairs can include but is notnecessarily limited to pairs such as S-protein and S-peptide, GST andGSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo Phe, KSI andoligo Leu, as well as “complementary” pairings such as oligo Arg witholigo Glu, and oligo Arg with oligo Asp. Thus, as used herein, thepeptide affinity group can comprise S-protein and S-peptide, GST andGSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI andoligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp. A morecomplete discussion of these binding pairs is included in U.S. Pat. No.7,183.392, incorporated herein by reference.

One member of the affinity binding pair will comprise part of a chimericconstruct joined to a nucleic acid while the corresponding member of thepair is affixed or immobilized to a matrix. It is also understood thateither member of a pair may be used as the non-nucleic acid portion suchthat it can be used with its corresponding member on the matrix. Thus,for instance, a chimeric nucleic acid can comprise an oligohistidineportion for capture by metal chelates attached to a solid matrix (anIMAC column or plate), or on the other hand, a nucleic acid can be usedthat has been modified by the presence of one or more metals allowingcapture on a matrix comprising oligohistidine or some other metalbinding peptide.

The chimeric nucleic acid provided by the present invention can compriseat least two portions, a first portion comprising a nucleic acidcomplementary to a nucleic acid sequence of interest, and a secondportion comprising a metal binding peptide, e.g., nickel, copper, cobaltor zinc. The metal binding peptide can be attached, desirably through alinker arm as previously described, to one or more nucleotides of thenucleic acid in the first portion through a sugar or phosphate of thenucleotide or nucleotides. As described elsewhere in this disclosure,the metal binding peptide can comprise any of the amino acid sequences:oligohistidine, HGGHHG, SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG,SPHHGGHPHHG, HPHHGGSPHHG, KDHLIHNVHKEEHAHAHNK, GHGLGHGHEQQHGLGHGHK orHGLGHGHEQQHGLGHGH. One or more energy transfer donors or one or moreenergy transfer acceptors can be incorporated into or attached to thechimeric nucleic acid just described.

Another chimeric nucleic acid provided by the present inventioncomprises at least two portions, a first portion comprising a nucleicacid complementary to a nucleic acid sequence of interest, and a secondportion comprising one member of a peptide affinity group. The membercan be attached, using a linker arm desirably, to one or morenucleotides of the nucleic acid in the first portion. As previouslydescribed, the peptide affinity binding group includes any of the pairs:S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptideand HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu,or oligo Arg and oligo Asp. This additional embodiment of a chimericnucleic acid can also further comprise one or more energy transferdonors, or one or more energy transfer acceptors.

Synthesis of the chimeric composition can be carried out by a variety ofmeans where either the base, sugar or phosphate position of a nucleotidein the nucleic acid portion is used to attach the affinity agent.Examples of means of modifying nucleic acids that may be used for thispurpose are described in Ward et al. in U.S. Pat. No. 4,711,955,Engelhardt et al., in U.S. Pat. No. 5,241,060, Stavrianopoulos et al.,in U.S. Pat. No. 4,707,440, Pergolizzi et al., in EP 0 611 828 andEngelhardt et al., in U.S. Patent Application No. 20030104620, all ofwhich are incorporated by reference. Attachment can be by means of acovalent attachment of one of the foregoing metals, oligopeptides orproteins to the nucleic acid portion, or it may be by means ofnoncovalent attachment through a secondary binding pair such as avidinand biotin. As an example of the latter, one of the proteins describedabove as a member of an affinity pair can be biotinylated using standardmethods and the nucleic acid can be covalently linked to strapavidin.Formation of a complex between these two entities will create chimericmolecule comprising the affinity member and a nucleic acid portion.

Covalent attachment may be direct where the affinity agent is attachedby itself to the nucleic acid portion or it may involve indirectcovalent attachment where there is a linker arm joining the affinityagent to the nucleic acid portion. The position of attachment of thenon-nucleotide portion to the nucleic acid can involve any chosennucleotide; i.e., either internal or terminal nucleotides are suitablefor carrying out the present invention. Linker arms are well-known inthe art and have been described by a number of authors and researchers.See, for example, Ward et al. in U.S. Pat. No. 4,711,955, Engelhardt etal., in U.S. Pat. No. 5,241,060, Engelhardt et al., in U.S. Pat. No.4,894,325, and Stavrianopoulos et al., in U.S. Pat. No. 7,186,478, allof which are incorporated herein by reference.

By means of the present invention, the presence of the nucleic acidportion will allow the capture of a nucleic acid and binding of it to amatrix through the affinity agent. The species of interest can be asbroad or as narrow as the user desires by the appropriate choice ofsequences used for the nucleic acid portion. For instance, the sequencecan be selective for a single species such as a nucleic acid coding fora particular gene, or it may represent an entire class of molecules.Selectivity can be carried out with a single sequence in a chimericcomposition or there may be more than one selective sequence in thechimeric composition. It is also envisioned that selectivity fordifferent sequences may be carried out either sequentially or inparallel by having different selective sequences as part of separatechimeric compositions.

As such, sequences in the nucleic acid portion can comprise genericsequences such as oligo T or oligo A that can bind to a wide variety ofdifferent nucleic acids or the nucleic acid portion may comprise uniquesequences that will bind to specific mRNA or cDNA species. Anillustration of a possible means of carrying this out is shown in FIG.1A. This aspect of the present invention may be used for either positiveor negative selection. As an example of positive selection, the nucleicacid portion may comprise oligo or poly T sequences allowing thesubsequent binding of polyA mRNA. Since mRNA generally consists of only3-6% of total RNA, the subsequent removal of RNA unable to bind to amatrix bound chimeric construct results in a powerful enrichment of thepoly A sequences that may be then used for a variety of purposes. As anexample of negative selection, the majority of total RNA consists ofrRNA sequences and these may be removed by the use of chimeric moleculesthat comprises sequences complementary to rRNA. After binding ofcomplexes to a matrix, the portion of the total RNA that contains mRNA,hnRNA, μRNA and snRNA remains unbound, thereby allowing any and all ofthese species to be used in further steps. This may be of special useand significance when the foregoing analytes are desirable as labelednucleic acids and the rRNA itself is of no use or interest. In suchcases, the presence of the rRNA may even be deleterious since it mayconsume reagents and contribute noise to analytic methods, as seen forexample, when total RNA is labeled by photobiotin, 94-97% of the labeledmaterial would be irrelevant to analysis of polyA mRNA.

The nucleic acids of the present invention may also be used in a numberof different ways: as part of a detection system; where a label may beincluded as part of the composition itself; when the analyte is beingdetected or quantified; or when a probe recognizes the analyte orcombinations thereof. For instance, nucleic acid analytes frombiological samples may be labeled directly by modifying the base, sugaror phosphate moieties. On the other hand, analytes may also be labeledduring the course of copying or in amplification procedures wherelabeled nucleotides are provided during the course of such procedures,thereby synthesizing labeled complementary or identical copies of theoriginal analytes. A chimeric composition could be used as a primer togenerate a labeled complementary copy that may be subsequently isolatedafterwards by means of the second member of the affinity pair or anormal primer could be used preparation of the labeled complementarycopies where a hybridization with a chimeric composition is carried outafterwards. An example of a copying reaction that may find use in thepresent invention could be the use of samples containing mRNA wherelabeled cDNA copies are prepared by means of reverse transcriptase or aDNA polymerase with reverse transcriptase activity. A general depictionof this type of format is shown in FIG. 1B.

In principle, the same methods can be applied to amplification reactionswhere there are a series of copying reactions. Examples of amplificationsystems that may be useful in the present invention can include but arenot necessarily limited to the polymerase chain reaction (PCR), ligasechain reaction (LCR), transcription mediated amplification (TMA), Stranddiplacement amplification (SDA), Nucleic acid sequence basedamplification (NASBA) and Secondary Structure Amplification (Rabbani etal., in U.S. Pat. No. 6,743,605) all of which are incorporated byreference. Amplifications may be directed towards specific nucleic acidsequences as is generally used in the preceding methods, or there may bea more global amplification of multiple sequences from a library thatincludes the preceding methods as well as methods such as those taughtby Van Gelder et al., in U.S. Pat. No. 5,545,522, Kurn in U.S. Pat. No.6,251,639 and Stavrianopoulos et al., in U.S. Pat. No. 7,163,796, all ofwhich are incorporated by reference. The synthesis of nucleic acids maytake place after the nucleic acid(s) of interest have been isolated froma biological sample and released from a matrix or the reactions may takeplace while the nucleic acids are still bound to the matrix. In thelatter case, the nucleic acids of the present invention may be used in apassive manner where they are only used to immobilize a nucleic acid inan environment where nucleic acid synthesis reactions may take place.Alternatively, it may be an active participant where the chimericnucleic acid comprises a promoter or acts as a primer in reactions suchas those cited above.

Detection of an analyte may also take place with unlabled analytes bymeans of the additional use of a labeled probe that is complementary tothe nucleic acid(s) of interest. This may be used with nucleic acids intheir native forms, or complementary copies derived form copying oramplification procedures. A depiction of a format with this process isshown in FIG. 1C.

Other formats are also possible involving energy transfer elements whereeither a capture nucleic acid, an analyte or a probe is labeled with oneor more energy transfer donors and one of the foregoing is labeled withan energy acceptor. Examples of various formats that could be used withthis arrangement are shown in FIGS. 1D-1F. Thus, the compositions of thethe present invention can comprise one or more energy transfer donors,or one or more energy transfer acceptors.

The present invention and the above-described compositions can be usedto isolate one or more species of a nucleic acid of interest. In onesuch process, various elements would be provided including a samplecontaining or suspected of containing the nucleic acid of interest, acomposition which comprises a nucleic acid portion and a first member ofan affinity binding pair, wherein the nucleic acid portion comprisessequences complementary to the nucleic acid species of interest, and theaffinity binding pair comprises (a) a metal binding peptide and animmobilized metal, or (b) a peptide affinity group; and wherein thefirst member of the affinity binding pair being attached, for example,through a linker arm, to one or more nucleotides in said nucleic acidportion; and a matrix comprising a second member of the affinity bindingpair. In this process, the composition hybridizes with any nucleic acidof interest contained in the sample to form a first complex. This isfollowed by contacting the first complex with the matrix provided toform a second complex by means of a binding interaction between thefirst member and the second member of the affinity binding pair. Thematerial bound to the matrix could then be separated from unboundmaterial, thereby isolating said nucleic acid species of interest. Thus,the portion of the sample that remains bound to the matrix can or maycomprise the nucleic acid species of interest. Contrariwise, the portionof the sample that remains unbound to the matrix may or could comprisethe nucleic acid species of interest. It should be understood to thoseskilled in the art that one or more washing steps could be used in theprocess just described above. The metal binding peptide, the immobilizedmetal, the peptide affinity group, linker arms, have been describedabove with respect to other descriptions of the present compositions andprocesses.

In a different application of the present invention, a process isprovided for detecting the presence or quantity of a nucleic acid ofinterest. In this detection or quantification process, various elementsare provided. These include a sample containing labeled nucleic acids, acomposition comprising a nucleic acid portion and a first member of anaffinity binding pair, wherein the nucleic acid portion comprisessequences complementary to the nucleic acid of interest, and a solidsupport or matrix comprising a second member of the affinity bindingpair. The affinity binding pair can comprise: (a) a metal bindingpeptide and an immobilized metal, or (b) a peptide affinity group. Thefirst member of the affinity binding pair is attached to one or morenucleotides of said nucleic acid portion, and this attachment can bethrough a linker arm as described in further detail above. Using theelements provided, the above composition is hybridized with any labelednucleic acid of interest contained in the sample to form a firstcomplex. This first complex is contacted with the matrix to form asecond complex by means of a binding interaction between the firstmember and the second member of the affinity binding pair. The matrix iswashed one or more times to remove unhybridized nucleic acids from thematrix. Detection or quantification of the nucleic acid of interest iscarried out by means of detecting or quantifying a signal from thelabels. Such labels are detectable fluorescently, chemiluminescently,colorimetrically or enzymatically. The just described process caninclude a further step of releasing the second complex from the matrixprior to detecting or quantifying the nucleic acid of interest. Thenature of the metal binding peptide, the immobilized metal, the peptideaffinity group, the linker arm, energy transfer donors and energytransfer acceptors have been described earlier in this disclosure andneed not be reiterated here.

Other processes for detecting or quantifying nucleic acids of interestare also contemplated and provided by this invention. In one suchdetection or quantification process, the following elements areprovided: a sample containing or suspected of containing the nucleicacid of interest; a labeled probe complementary to the nucleic acid ofinterest; a composition comprising a nucleic acid portion and a firstmember of an affinity binding pair, wherein the nucleic acid portioncomprises sequences complementary to the nucleic acid of interest,wherein the affinity binding pair comprises (a) a metal binding peptideand an immobilized metal, or (b) a peptide affinity group, and whereinthe first member of the affinity binding pair is attached, e.g., througha linker arm, to one or more nucleotides of said nucleic acid portionthrough a sugar, phosphate or base of the nucleotide or nucleotides; anda matrix comprising a second member of the affinity binding pair. Inthis process, any nucleic acids of interest in the sample are hybridizedwith labeled probe and the composition to form a first complex. Thisfirst complex is contacted with the matrix to form a second complex bymeans of a binding interaction between the first member and the secondmember of the affinity binding pair. The matrix can be washed one ormore times to remove unbound materials from the sample. Detection orquantification of the nucleic acid of interest can be carried out bymeans of detecting or quantifying a signal from the labels. Such labelsare detectable fluorescently, chemiluminescently, colorimetrically orenzymatically. An additional step of releasing the second complex fromthe matrix can be carried out or included in this process prior tocarrying out detection or quantification.

In the process just described above, aspects such as the metal bindingpeptide, the immobilized metal, the peptide affinity group, the linkerarm, energy transfer donors, energy transfer acceptors, and the like,have been described previously in this disclosure and will not bereiterated. With respect to the energy transfer elements, it should beunderstood that the labeled probes can comprise one or more energytransfer donors and the composition can comprise one or more energytransfer acceptors. Alternatively, the labeled probes can comprise oneor more energy transfer acceptors and the composition can comprise oneor more energy transfer donors. As a different variation, the labeledprobes can comprise one or more energy transfer donors and the nucleicacids in the sample provided can be labeled with one or more energytransfer acceptors. Alternatively, in this different variation, thelabeled probes can comprise one or more energy transfer acceptors andthe nucleic acids in the sample provided can be labeled with one or moreenergy transfer donors.

In a different embodiment, the present invention and compositions can bedirected to another process for detecting the presence or quantity of anucleic acid of interest. Initially provided are several elementsincluding: a sample containing or suspected of containing the nucleicacid of interest; a probe complementary to the nucleic acid of interestand comprising two portions, wherein a first comprises sequencescomplementary to the nucleic acid of interest, and a second portioncomprising a signal sequence; a composition comprising a nucleic acidportion and a first member of an affinity binding pair, wherein thenucleic acid portion comprises sequences complementary to the nucleicacid of interest, wherein the affinity binding pair comprises (a) ametal binding peptide and an immobilized metal, or (b) a peptideaffinity group, and wherein the first member of the affinity bindingpair is attached, through a linker arm, for example, to one or morenucleotides of the nucleic acid; and a matrix comprising a second memberof the affinity binding pair. Any nucleic acids of interest which are inthe sample are allowed to hybridize with the labeled probe and thecomposition to form a first complex. Such first complex is contactedwith the matrix to form a second complex by means of bindinginteractions between one or more binding partners and the affinitypeptide. The matrix is washed in a single step or a series of washingsteps to remove unbound materials from the sample. Detection orquantification of the nucleic acid of interest is carried out byhybridizing labeled oligonucleotides complementary to the signalsequence. The labeled oligonucleotides are detectable fluorescently,chemiluminescently, colorimetrically or enzymatically. The nature of themetal binding peptide, i.e., the amino acid sequences used therein, theimmobilized metal, the peptide affinity group, and the like, have beenpreviously described in this disclosure and will not be repeated here.

The signal sequence that may be used for this purpose have beendescribed previously, including methods and compositions described byPergolizzi et al., in European Publication No. 0 128 332 A1, based onU.S. patent application Ser. No. 06/491,929, filed May 5, 1983; andUrdea et al., U.S. Pat. No. 5,124,246. In this aspect of the presentinvention, such signal sequence can comprise a homopolymeric sequence,or it can comprise a heterologous sequence where the heterologoussequence is neither identical or complementary to the nucleic acid ofinterest.

In another aspect of the present invention, the use of the kininogenpeptide sequence is disclosed as being useful for incorporation intonucleic acids coding for proteins of interest. The provision of thisnovel affinity peptide may increase the range of fusion proteins thatmay be successfully designed with an affinity sequence. As mentionedearlier, even the flexibility of being able to use either the carboxy oramino terminus as an insertion site may be insufficient and bothlocations may interfere in either production or activity of therecombinant protein of interest. The availability of an alternativepeptide sequence may allow generation of recombinant proteins thatovercome this problem.

Other components or elements can be added to the just-describedcomposition including one or more energy transfer donors or one or moreenergy transfer acceptors.

As such, this invention is also directed to and provides a fusionprotein comprising a biologically active polypeptide or protein and atleast one affinity peptide attached to the amino-terminus or thecarboxyl-terminus of the biologically active polypeptide or protein,wherein the affinity peptide comprises at least a portion of the aminoacid sequence of kininogen, the portion comprising a metal bindingpeptide. In a different aspect, the invention also provides a fusionprotein comprising a biologically active polypeptide or protein and anaffinity peptide attached at the amino-terminus and an affinity peptideattached at the carboxyl-terminus of the biologically active polypeptideor protein, wherein the affinity peptides comprise at least a portion ofthe amino acid sequence kininogen, the portion also comprising a metalbinding peptide. A preferred sequence for the kininogen used as theaffinity peptide in such fusion proteins is GHGLGHGHEQQHGLGHGHK, or aportion thereof. The kininogen can be human kininogen if desired. Otheraspects of the fusion proteins just described above should be noted. Oneaspect relates to the amino acid sequence between the biologicallyactive polypeptide or protein and the affinity peptides, and thissequence is or can be recognizable by a protease, such as enterokinaseor coagulation factor X_(a). Further, the affinity peptide may bindnickel, copper, cobalt or zinc.

A format may be used where the affinity tagged antibody is specific fora unique target of interest where the target may be a protein or someother molecule of interest. This approach entails construction of aunique antibody for each antigen of interest and it has been previouslydescribed in the context of protein arrays by Wingren et al., (2005Proteomics 5; 1281-1291) where a library of single-chain Fv antibodieswere fixed to a matrix by either a metal or an anti-tag antibody. Otherantibodies that have been modified this way have been described byJohnson et al. in U.S. Patent Application No. 2004/0197866 and Wu etal., in U.S. Patent Application No. 2006/0094062.

It should be appreciated that the present invention can be used toprovide a process for modifying proteins of interest. To modify such aprotein, three elements are provided including: (i) a nucleic acid thatcodes for the protein of interest; (ii) a nucleic acid that codes for aportion of the amino acid sequence of kininogen, wherein the portioncodes for a metal binding peptide; and (iii) an expression vector. Tomodify the protein with the elements provided, the nucleic acid (ii) isadded to the nucleic acid (i) to generate a nucleic acid coding for afusion protein. The fusion protein coding nucleic acid is inserted intothe expression vector (iii), thereby generating a vector that expressesthe modified protein of interest. Other aspects of the just describedprotein modification process deserve mention. One aspect concerns theexpression vector (iii) and it can comprise a number of different types,including a mammalian expression vector, a bacterial expression vector,an insect cell expression vector and a yeast expression vector. Theexpression vector (iii) can be plasmid or a viral vector.

Thus, this invention when applied to the isolation of a protein ofinterest, provides the following process. Several elements are providedincluding (i) a nucleic acid that codes for the protein of interest;(ii) a nucleic acid that codes for a portion of the amino acid sequenceof kininogen, wherein that portion codes for a metal binding peptide;(iii)an expression vector; and (iv) a metal-modified matrix. To isolatethe protein of interest, the nucleic acid (ii) is added to the nucleicacid (i) to generate a nucleic acid coding for a fusion protein. Thenucleic acid coding for the fusion protein is inserted into theexpression vector (iii), resulting in the expression of the protein ofinterest. Purification can be carried out by binding the protein ofinterest to the metal-modified matrix (iv). This can be desirablyperformed using a chromatographic column or a microtitre plate as themetal-modified matrix. As described previously, the expression vector(iii) can comprise a mammalian expression vector, a bacterial expressionvector, an insect cell expression vector or a yeast expression vector.The expression vector (iii) can also be a plasmid or a viral vector.

In another embodiment of the present invention, a method of isolation ordetection of proteins is described. As described earlier, theincorporation of an amino sequence for an affinity tag has beenincorporated into proteins to effect an ease of isolation. However, thisentails a genetic modification of the protein of interest and it hasbecome clear that even with a flexibility of being able to add to eitherthe carboxy or the amino end, some proteins lose functionality by suchmeans. This system does not allow the detection of unaltered or nativeproteins. Accordingly, it is disclosed herein that an antibody to aprotein can be engineered to have an amino sequence that comprises anaffinity peptide, thus allowing capture of the antibody onto a solidmatrix as well as any complex formed between the modified antibody andits target. This will be of special use and significance when the targetis a protein that is desired to be isolated.

On the other hand, a more universal reagent can be made by constructionof a tagged antibody that has an affinity for other antibodies. Thus,for example, an anti-goat antibody that is derived from mouse cells canbe redesigned to comprise an affinity peptide and used to collectcomplexes that are made of goat antibodies that are bound to theirparticular analyte targets. This system uses a universal reagent in thatonly the anti-goat antibody needs to be modified and this reagent shouldbe able to recognize a wide variety of complexes formed between goatantibodies and their antigen targets. Thus, the need for individuallymodifying each antibody used for as an antibody/antigen pair isobviated. Although this method can subsequently be used to isolate theantigen target by appropriate release of the target, it is understoodthat the present invention may also be used in formats that are used todetect or quantify targets by means of immunoassays. It is understoodthat when the terms “antibody” or “antibodies” are used in the presentinvention, such terms include, without limitation, antibody fragments,single chain antibodies, and the like.

This invention further provides a fusion protein comprising an antibodyand at least one affinity peptide attached to the amino terminus or thecarboxyl terminus of the antibody, wherein the affinity peptidecomprises a metal binding peptide or is one member of a peptide affinitygroup. The antibody has an affinity for a different antibody in thiscase. A different embodiment provided by the present invention is afusion protein comprising an antibody and an affinity peptide attachedto the amino terminus and an affinity peptide attached to carboxylterminus of the antibody. The affinity peptide comprises a metal bindingpeptide or is one member of a peptide affinity group, wherein theantibody has an affinity for a different antibody. In the case of eitherfusion protein, the peptide affinity group can comprise S-protein andS-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI andoligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg andoligo Asp. Additionally, the metal binding peptide can compriseoligohistidine or an oligopeptide comprising any of the followingsequences: HGGHHG, SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG, SPHHGGHPHHG,HPHHGGSPHHG, KDHLIHNVHKEEHAHAHNK, GHGLGHGHEQQHGLGHGHK orHGLGHGHEQQHGLGHGH.

Processes for analyte isolation and detection or quantification are alsoprovided by the present invention. To isolate an analyte of interest,the following process can be used in accordance with this invention.Four elements are initially provided, including (i) a sample containingor suspected of containing the analyte of interest; (ii) a firstantibody having an affinity for the analyte; (iii) a fusion antibodycomprising: (a) an antibody and at least one affinity peptide attachedto the amino terminus or the carboxyl terminus of the antibody, whereinthe affinity peptide comprises a metal binding peptide or is one memberof a peptide affinity group, wherein the antibody has an affinity for adifferent antibody; or (b) an antibody and an affinity peptide attachedto the amino terminus and an affinity peptide attached to carboxylterminus of the antibody, wherein the affinity peptide comprises a metalbinding peptide or is one member of a peptide affinity group, whereinthe antibody has an affinity for a different antibody; and (iv) a matrixcomprising a metal or a second member of the affinity peptide group.

In this analyte isolation process, the sample is allowed to contact thefirst antibody, thereby forming a first complex between the firstantibody and any analyte present in the sample. The first complex isallowed to contact with the fusion antibody, thereby forming a secondcomplex between the first complex and the fusion antibody. The secondcomplex is contacted with the matrix to bind the second complex to thematrix. Unbound material is removed from the matrix, and the analyte ofinterest is released from the second complex, thereby isolating theanalyte of interest.

In this analyte isolation process, other aspects can be described. Forexample, the peptide affinity group can comprise S-protein andS-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI andoligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg andoligo Asp. Further, the metal binding peptide can compriseoligohistidine or an oligopeptide comprising any of the sequences:HGGHHG, SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG, SPHHGGHPHHG,HPHHGGSPHHG, KDHLIHNVHKEEHAHAHNK, GHGLGHGHEQQHGLGHGHK orHGLGHGHEQQHGLGHGH.

Analyte detection or quantification can also be carried out inaccordance with this invention. In a process for detecting orquantifying an analyte of interest, e.g., a protein or a polypeptide,the following elements are provided: (i) a labeled sample containing orsuspected of containing labeled analyte of interest; (ii) a firstantibody having an affinity for the analyte; (iii) a fusion antibodycomprising: (a) an antibody and at least one affinity peptide attachedto the amino terminus or the carboxyl terminus of the antibody, whereinthe affinity peptide comprises a metal binding peptide or is one memberof a peptide affinity group, wherein the antibody has an affinity for adifferent antibody; or (b) an antibody and an affinity peptide attachedto the amino terminus and an affinity peptide attached to carboxylterminus of the antibody, wherein the affinity peptide comprises a metalbinding peptide or is one member of a peptide affinity group, whereinthe antibody has an affinity for a different antibody; and (iv) a matrixcomprising a metal or a second member of the affinity peptide group. Thesample is contacted with the first antibody, thereby forming a firstcomplex between the first antibody and any analyte present in thesample. The first complex so formed is contacted with the fusionantibody, thereby forming a second complex between the first complex andthe fusion antibody. The second complex is contacted with the matrix tobind the second complex to the matrix. Unbound material is removed fromthe matrix. Detection or quantification of the labeled analytes bound tothe matrix is performed by means of detecting or quantifying a signalfrom the labels. The labels are detectable fluorescently,chemiluminescently, colorimetrically or enzymatically. It should also benoted that an additional step can be performed in connection with thisprocess, namely, the analyte of interest can be released from the secondcomplex prior to performing any detection or quantification. Aspreviously described, the peptide affinity group can comprise S-proteinand S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSIand oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Argand oligo Asp. Further, the metal binding peptide can compriseoligohistidine or an oligopeptide comprising any of the sequences:HGGHHG, SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG, SPHHGGHPHHG,HPHHGGSPHHG, KDHLIHNVHKEEHAHAHNK, GHGLGHGHEQQHGLGHGHK orHGLGHGHEQQHGLGHGH.

This invention also provides a process for detecting and quantifying ananalyte of interest, such as a protein or polypeptide. In such aprocess, the following elements are provided: (i) a labeled samplecontaining or suspected of containing labeled analytes of interest; (ii)a first antibody having an affinity for the analyte; (iii) a fusionantibody comprising: (a) an antibody and at least one affinity peptideattached to the amino terminus or the carboxyl terminus of the antibody,wherein the affinity peptide comprises a metal binding peptide or is onemember of a peptide affinity group, wherein the antibody has an affinityfor a different antibody; or (b) an antibody and an affinity peptideattached to the amino terminus and an affinity peptide attached tocarboxyl terminus of the antibody, wherein the affinity peptidecomprises a metal binding peptide or is one member of a peptide affinitygroup, wherein the antibody has an affinity for a different antibody;and (iv) a matrix comprising a metal or a second member of the affinitypeptide group. In this detection or quantification process, a firstcomplex is formed among the matrix, the fusion antibody and the firstantibody. The first complex is contacted with the labeled sample to forma second complex between the first complex and any labeled analytes thatmay be present in the sample. Unbound material is removed from thematrix. Labeled analytes bound to the matrix are detected or quantifiedby means of detection or quantification of the the signal generated fromthe labels. Such labels are detectable fluorescently,chemiluminescently, colorimetrically or enzymatically. Furthermore, thematrix can comprise an array of different first antibodies, therebyallowing for detection or quantification of multiple analytes ofinterest.

The examples which follow are set forth to illustrate various aspects ofthe present invention but are not intended in any way to limit its scopeas more particularly set forth and defined in the claims that followthereafter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following are examples illustrating the present invention.

EXAMPLE 1 Preparation of Oligonucleotide Modified with OligohistidineStep 1 Synthesis of N-trifluoroheptahistidine-NHS Ester

32 mg (˜30 Moles) of acetylated heptahistidine (Biopeptides, Inc. SanDiego Calif.) were dissolved in 200 I methanol followed by addition of400 I of methyltrifluoroacetate and 50 I of pyridine and the mixtureleft overnight at room temperature. The liquid phase was evaporated by astream of argon and then evaporated in vacuo overnight to remove anytraces of pyridiniumtrifluoroacetate formed by the presence oftrifluoroacetic acid contaminants in the methyltrifluoroacetate. Theresidue was dissolved in 200 I of dimethylformamide (DMF) followed bythe addition of 60 moles of n-hydroxysuccinimide and then 50 I of 0.9 Mdicyclohexylcarbodiimide in DMF. The mixture was stirred overnight and aurea precipitate was removed by centrifugation.

Step 2 Preparation of Amine Modified Rhodamine Labeled Oligonucleotide

A 5′ amino modified oligonucleotide was ordered from Sigma Genosys(Sigma-Aldrich, St. Louis, Mo.) with the following sequence: 5′amine-tcaaccaac 3′. The oligonucleotide was labeled by terminaltransferase using a 3′-Oligonucleotide labeling system (Enzo LifeSciences Inc, Farmingdale, N.Y.) and rhodamine labeled dUTP (Enzo LifeSciences Inc, Farmingdale, N.Y.).

Step 3 Addition of the Oligohistidine Peptide to the Rhodamine LabeledOligonucleotide.

100 g of the the oligo prepared in step 2 was phenol extracted, ethanolprecipitated and dissolved in 200 l of 0.2M Sodium Borate, 5 mM EDTA, pH8.5 followed by addition of 300 I of DMF and 50 I of theN-trifluoroheptahistidine-NHS ester synthesized in step 1. The mixturewas stirred overnight and the derivatized DNA was then precipitated bythe addition of 10 volumes of n-butanol. The pellet was dissolved in 200ul of 1M Lithium Hydroxide solution and left at room temperature for 30minutes to remove the trifluoroacetyl groups. The heptahistidinemodified DNA was then precipitated with 10 volumes of ethanol andredissolved in binding buffer (20 mM Phosphate, 500 mM NaCl, pH 7.4)

EXAMPLE 2 Efficiency of Binding and Release of Oligohisitidine Modifiedoligonucleotide to Nickel coated sepharose beads (Ni-Column)

The heptahistidine modified rhodamine oligonucleotide from Example 1 wasdiluted in binding buffer (20 mM Phosphate, 500 mM NaCl, pH 7.4) andadded to a Ni-column (Ni Sepharose high performance, GE Healthcare,Piscataway, N.J.). As a control, rhodamine labeled oligonucleotidewithout the addition of the heptahistidine was also added to aNi-column. The columns were then washed with 8 volumes of binding bufferfollowed by a 5 volumes of binding buffer containing 0.5 M Imidazole torelease the oligonucleotide histidine groups and the eluants collected.Quantification was carried out using a spectrofluorometer (Ex: 556 nm,Em: 580) for both the Effluent (Ft) that did not bind and for the Eluent(Elu) that was released after binding.

The results of this experiment are shown in FIG. 2(A) as represented bythe percentage of the rhodamine signal of the input material. In thisexperiment 75% of the oligohistidine modified oligonucleotide bound tothe column while only 8% of the unmodified rhodamine oligonucleotideremained bound. The lack of quantitative binding by the oligohistidinemodified preparation is likely to be an indication that not all of theoligonuclotides were conjugated to the peptide. This was confirmed bytaking the effluent that was unable to bind and running it a second timeover the Ni-column where the level of binding was observed to be thesame as previously observed with the oligonucleotide lacking thehisitidine (data not shown).

EXAMPLE 3 Efficiency of Binding of Oligohistidine ModifiedOligonucleotide to Nickel Coated 96-Well Plates.

Binding of the oligohistidine modified oligonucleotide to a matrix wasalso tested by binding to 96 well plates instead of the columns used inExample 2. Identical dilutions of histidine modified and unmodifiedrhodamine labeled oligonucleotides from Example 2 were added toNickel-coated plates (HisGrab Nickel coated 96-well plates, Pierce,Rockford, Ill.) and incubated for 3 hours at room temperature, followedby washing 3 times with 200 ul binding buffer. The bound DNA wasmeasured by detecting rhodamine with a plate reader (filters: Ex550,Em610, Fluostar Optima, BMG Labtech). Results of this experiment areshown in FIG. 2(B). The Histidine-modified DNA bound to the Ni-platesmore effectively than the control DNA. For example, an input of 10 ul ofcontrol and His(7)-DNA showed 90% of the histidine modified DNA beingbound to the wells, while only 10% of the control DNA was detected. Theresults shown in FIG. 2 (B) also indicate that with the highest inputlevel (50 ul), the wells were overloaded.

EXAMPLE 4 Effects of Other Reagents on Binding

Preparations of nucleic acids are commonly taken up in the presence ofchelators such as EDTA or SSC (standard saline citrate). Since it ispossible that these could be competitors for a peptide/chelateinteraction, the oligonucleotides from Example 1 were tested for theability to be bound in their presence. The histidine modified oligoswere incubated with Ni beads in the presence of binding buffer(control), or binding buffer with either 0.5 mM EDTA or 1×SSC . Theresults of this Experiment are shown in FIG. 3, where it can be seenthat the presence of at least low levels of these components had noeffect on the efficiency of binding of the oligohistidine modifiednucleic acid.

Many obvious variations will be suggested to those of ordinary skill inthe art in light of the above detailed descriptions of the presentinvention. All such obvious variations are fully contemplated and areembraced by the scope and spirit of the present invention as set forthin the claims that now follow.

1. A composition which comprises a nucleic acid and one member of an affinity binding pair, wherein said member is attached to one or more nucleotides of said nucleic acid through a phosphate or sugar of said nucleotide or nucleotides.
 2. The composition of claim 1, wherein said affinity binding pair comprises: (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group.
 3. The composition of claim 2, wherein said metal binding peptide comprises any of the amino acid sequences: oligohistidine, HGGHHG (SEQ ID NO: 1), SPHHG (SEQ ID NO: 2), SPHHGGSPHHG (SEQ ID NO: 3), HPHHG (SEQ ID NO: 4), HPHHGGHPHHG (SEQ ID NO: 5), SPHHGGHPHHG (SEQ ID NO: 6), HPHHGGSPHHG (SEQ ID NO: 7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10) or HGLGHGHEQQHGLGHGH (SEQ ID NO: 9).
 4. The composition of claim 2, wherein said immobilized metal comprises nickel, copper, cobalt or zinc.
 5. The composition of claim 2, wherein said peptide affinity group comprises S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
 6. The composition of claim 1, wherein said member is attached to said nucleotide or nucleotides through a linker arm.
 7. The composition of claim 1, further comprising one or more energy transfer donors.
 8. The composition of claim 1, further comprising one or more energy transfer acceptors.
 9. A chimeric nucleic acid comprising at least two portions, a first portion comprising a nucleic acid complementary to a nucleic acid sequence of interest, and a second portion comprising a metal binding peptide, wherein said metal binding peptide is attached to one or more nucleotides of the nucleic acid in said first portion through a sugar or phosphate of said nucleotide or nucleotides.
 10. The composition of claim 9, wherein said metal binding peptide comprises any of the amino acid sequences: oligohistidine, HGGHHG (SEQ ID NO: 1), SPHHG (SEQ ID NO: 2), SPHHGGSPHHG (SEQ ID NO: 3), HPHHG (SEQ ID NO: 4), HPHHGGHPHHG (SEQ ID NO: 5), SPHHGGHPHHG (SEQ ID NO: 6), HPHHGGSPHHG (SEQ ID NO: 7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10) or HGLGHGHEQQHGLGHGH (SEQ ID NO: 9).
 11. The composition of claim 9, wherein said metal or metals are attached to said nucleotide or nucleotides through a linker arm.
 12. The composition of claim 9, wherein said metal binding peptide has an affinity for nickel, copper, cobalt or zinc.
 13. The composition of claim 9, further comprising one or more energy transfer donors.
 14. The composition of claim 9, further comprising one or more energy transfer acceptors.
 15. A chimeric nucleic acid comprising at least two portions, a first portion comprising a nucleic acid complementary to a nucleic acid sequence of interest, and a second portion comprising one member of a peptide affinity group, wherein said member is attached to one or more nucleotides of said nucleic acid in said first portion.
 16. The chimeric nucleic acid of claim 15, wherein said peptide affinity binding group comprises any of the pairs: S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
 17. The composition of claim 15, wherein said member is attached to said nucleotide or nucleotides through a linker arm.
 18. The composition of claim 15, further comprising one or more energy transfer donors.
 19. The composition of claim 15, further comprising one or more energy transfer acceptors.
 20. A process for isolating one or more species of a nucleic acid of interest, comprising the steps of: providing: a sample containing or suspected of containing said nucleic acid of interest, a composition which comprises a nucleic acid portion and a first member of an affinity binding pair, wherein said nucleic acid portion comprises sequences complementary to said nucleic acid species of interest, wherein said affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group; and wherein said first member of the affinity binding pair is attached to one or more nucleotides in said nucleic acid portion; and a matrix comprising a second member of said affinity binding pair; hybridizing said composition with any nucleic acid of interest contained in said sample to form a first complex; contacting said first complex with said matrix to form a second complex by means of a binding interaction between said first member and said second member of the affinity binding pair; and separating bound from unbound material, thereby isolating said nucleic acid species of interest.
 21. The process of claim 20, wherein said metal binding peptide comprises any of the amino acid sequences: oligohistidine, HGGHHG (SEQ ID NO: 1), SPHHG (SEQ ID NO: 2), SPHHGGSPHHG (SEQ ID NO: 3), HPHHG (SEQ ID NO: 4), HPHHGGHPHHG (SEQ ID NO: 5), SPHHGGHPHHG (SEQ ID NO: 6), HPHHGGSPHHG (SEQ ID NO: 7). KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10) or HGLGHGHEQQHGLGHGH (SEQ ID NO: 9).
 22. The process of claim 20, wherein said immobilized metal comprises nickel, copper, cobalt or zinc.
 23. The process of claim 20, wherein said peptide affinity group comprises S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
 24. The process of claim 20, wherein said first member of said affinity binding pair is attached to said one or more nucleotides through a linker arm.
 25. The process of claim 20, wherein in said separating step the portion of said sample that remains unbound to said matrix comprises said nucleic acid species of interest.
 26. The process of claim 20, wherein the portion of said sample that remains bound to said matrix comprises said nucleic acid species of interest.
 27. The process of claim 20, further comprising one or more washing steps.
 28. A process for detecting the presence or quantity of a nucleic acid of interest, comprising the steps of: providing: a sample containing labeled nucleic acids, a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein said nucleic acid portion comprises sequences complementary to said nucleic acid of interest, and a matrix comprising a second member of said affinity binding pair; wherein said affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group; and wherein said first member of the affinity binding pair is attached to one or more nucleotides of said nucleic acid portion; and hybridizing said composition with any nucleic acid of interest contained in said sample to form a first complex; contacting said first complex with said matrix to form a second complex by means of a binding interaction between said first member and said second member of said affinity binding pair; and washing said matrix to remove unhybridized nucleic acids from said matrix; and detecting or quantifying said nucleic acid of interest by means of detecting or quantifying a signal from said labels.
 29. The process of claim 28, wherein said metal binding peptide comprises any of the amino acid sequences: oligohistidine, HGGHHG (SEQ ID NO: 1), SPHHG (SEQ ID NO: 2), SPHHGGSPHHG (SEQ ID NO: 3), HPHHG (SEQ ID NO: 4), HPHHGGHPHHG (SEQ ID NO: 5), SPHHGGHPHHG (SEQ ID NO: 6), HPHHGGSPHHG (SEQ ID NO: 7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10) or HGLGHGHEQQHGLGHGH (SEQ ID NO: 9).
 30. The process of claim 28, wherein said immobilized metal comprises nickel, copper, cobalt or zinc.
 31. The process of claim 28, wherein said peptide affinity group comprises S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
 32. The process of claim 28, wherein said first member of the affinity binding pair is attached to said nucleotide or nucleotides through a linker arm.
 33. The process of claim 28, wherein said labeled nucleic acids in the sample comprise one or more energy transfer donors and wherein said composition comprise one or more energy transfer acceptors.
 34. The process of claim 28, wherein said labeled nucleic acids in the sample comprise one or more energy transfer acceptors and wherein said composition comprise one or more energy transfer donors.
 35. The process of claim 28, wherein said labeled nucleic acids comprise labels that are fluorescently, chemiluminescently, colorimetrically or enzymatically detectable.
 36. The process of claim 28, further comprising a step of releasing said second complex from said matrix prior to said detecting step.
 37. A process for detecting the presence or quantity of a nucleic acid of interest, comprising the steps of: providing: a sample containing or suspected of containing said nucleic acid of interest; a labeled probe complementary to said nucleic acid of interest; a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein said nucleic acid portion comprises sequences complementary to said nucleic acid of interest, wherein said affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group, and wherein said first member of the affinity binding pair is attached to one or more nucleotides of said nucleic acid portion through a sugar, phosphate or base of said nucleotide or nucleotides; and a matrix comprising a second member of said affinity binding pair; hybridizing any nucleic acids of interest in said sample with labeled probe and said composition to form a first complex; contacting said first complex with said matrix to form a second complex by means of a binding interaction between said first member and said second member of said affinity binding pair; washing said matrix to remove unbound materials from said sample; and detecting or quantifying said nucleic acid of interest by means of detecting or quantifying a signal from said labels.
 38. The process of claim 37, wherein said metal binding peptide (a) comprises any of the amino acid sequences: oligohistidine, HGGHHG (SEQ ID NO: 1), SPHHG (SEQ ID NO: 2), SPHHGGSPHHG (SEQ ID NO: 3), HPHHG (SEQ ID NO: 4), HPHHGGHPHHG (SEQ ID NO: 5), SPHHGGHPHHG (SEQ ID NO: 6), HPHHGGSPHHG (SEQ ID NO: 7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10) or HGLGHGHEQQHGLGHGH (SEQ ID NO: 9).
 39. The process of claim 37, wherein said immobilized metal comprises nickel, copper, cobalt or zinc.
 40. The process of claim 37, wherein said peptide affinity group comprises S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
 41. The process of claim 37, wherein said first member of the affinity binding pair is attached to said nucleotide or nucleotides through a linker arm.
 42. The process of claim 37, wherein said labeled probes comprise one or more energy transfer donors and wherein said composition comprises one or more energy transfer acceptors.
 43. The process of claim 37, wherein said labeled probes comprise one or more energy transfer acceptors and wherein said composition comprises one or more energy transfer donors.
 44. The process of claim 37, wherein said labeled probes comprise one or more energy transfer donors and wherein the nucleic acids in said sample have been labeled with one or more energy transfer acceptors.
 45. The process of claim 37, wherein said labeled probes comprise one or more energy transfer acceptors and wherein the nucleic acids in said sample have been labeled with one or more energy transfer donors.
 46. The process of claim 37, wherein said labeled nucleic acids are detected fluorescently, chemiluminescently, colorimetrically or enzymatically.
 47. The process of claim 37, further comprising a step of releasing said second complex from said matrix prior to said detecting step.
 48. A process for detecting the presence or quantity of a nucleic acid of interest, comprising the steps of: providing: a sample containing or suspected of containing said nucleic acid of interest; a probe complementary to said nucleic acid of interest and comprising two portions, wherein a first comprises sequences complementary to said nucleic acid of interest, and a second portion comprising a signal sequence; a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein said nucleic acid portion comprises sequences complementary to said nucleic acid of interest, wherein said affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group, and wherein said first member of the affinity binding pair is attached to one or more nucleotides of said nucleic acid; and a matrix comprising a second member of said affinity binding pair; hybridizing any nucleic acids of interest in said sample with labeled probe and said composition to form a first complex; contacting said first complex with said matrix to form a second complex by means of a binding interaction between said one or more binding partners and said affinity peptide; washing said matrix to remove unbound materials from said sample; and detecting or quantifying said nucleic acid of interest by hybridizing labeled oligonucleotides complementary to said signal sequence.
 49. The process of claim 48, wherein said metal binding peptide comprises any of the amino acid sequences oligohistidine, HGGHHG (SEQ ID NO: 1), SPHHG (SEQ ID NO: 2), SPHHGGSPHHG (SEQ ID NO: 3), HPHHG (SEQ ID NO: 4), HPHHGGHPHHG (SEQ ID NO: 5), SPHHGGHPHHG (SEQ ID NO: 6), HPHHGGSPHHG (SEQ ID NO: 7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10) or HGLGHGHEQQHGLGHGH (SEQ ID NO: 9).
 50. The process of claim 48, wherein said immobilized metal comprises nickel, copper, cobalt or zinc.
 51. The process of claim 48, wherein said peptide affinity group comprises S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
 52. The process of claim 48, wherein said labeled oligonucleotides are detected fluorescently, chemiluminescently, colorimetrically or enzymatically.
 53. The process of claim 48, wherein said signal sequence comprises a homopolymeric sequence.
 54. The process of 48, wherein said signal sequence comprises a heterologous sequence, wherein said heterologous sequence is neither identical or complementary to said nucleic acid of interest.
 55. A fusion protein comprising a biologically active polypeptide or protein and at least one affinity peptide attached to the amino-terminus or the carboxyl-terminus of said biologically active polypeptide or protein, wherein said affinity peptide comprises at least a portion of the amino acid sequence of kininogen, said portion comprising a metal binding peptide.
 56. A fusion protein comprising a biologically active polypeptide or protein and an affinity peptide attached at the amino-terminus and an affinity peptide attached at the carboxyl-terminus of said biologically active polypeptide or protein, wherein said affinity peptides comprise at least a portion of the amino acid sequence kininogen, said portion comprising a metal binding peptide.
 57. The fusion protein of claim 55 or 56, wherein said affinity peptide comprises the sequence GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10), or a portion thereof.
 58. The fusion protein of claim 55 or 56, wherein said kininogen comprises human kininogen.
 59. The fusion protein of claim 55 or 56, wherein said fusion protein comprises an amino acid sequence between said biologically active polypeptide or protein and said affinity peptides, wherein said sequence is recognizable by a protease.
 60. The fusion protein of claim 59, wherein said protease comprises enterokinase or coagulation factor X_(a).
 61. The fusion protein of claim 55, wherein said affinity peptide binds nickel, copper, cobalt or zinc.
 62. A fusion protein comprising an antibody linked by its amino- and/or carboxyl-terminus to one or two affinity peptides, wherein said affinity peptide binds to a metal, and wherein said antibody has an affinity to an epitope on a different antibody.
 63. The fusion protein of claim 62, wherein said affinity peptide comprises oligohistidine or an oligopeptide comprising the sequence HGGHHG (SEQ ID NO: 1), SPHHG (SEQ ID NO: 2), SPHHGGSPHHG (SEQ ID NO: 3), HPHHG (SEQ ID NO: 4), HPHHGGHPHHG (SEQ ID NO: 5), SPHHGGHPHHG (SEQ ID NO: 6), HPHHGGSPHHG (SEQ ID NO: 7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10) or HGLGHGHEQQHGLGHGH (SEQ ID NO: 9).
 64. A process for modifying a protein of interest, said process comprising the steps of: providing (i) a nucleic acid that codes for said protein of interest; (ii) a nucleic acid that codes for a portion of the amino acid sequence of kininogen, wherein said portion codes for a metal binding peptide; and (iii) an expression vector; adding said nucleic acid (ii) to said nucleic acid (i) to generate a nucleic acid coding for a fusion protein; and inserting said nucleic acid coding for said fusion protein into said expression vector (iii), thereby generating a vector that expresses said modified protein of interest.
 65. The process of claim 64, wherein said expression vector (iii) comprises a mammalian expression vector, a bacterial expression vector, an insect cell expression vector and a yeast expression vector.
 66. The process of claim 64, wherein said expression vector (iii) comprises a plasmid or viral vector.
 67. A process for isolating a protein of interest, said process comprising the steps of: providing: (i) a nucleic acid that codes for said protein of interest; (ii) a nucleic acid that codes for a portion of the amino acid sequence of kininogen, wherein said portion codes for a metal binding peptide; (iii) an expression vector; and (iv) a metal-modified matrix; adding said nucleic acid (ii) to said nucleic acid (i) to generate a nucleic acid coding for a fusion protein; inserting said nucleic acid coding for said fusion protein into said expression vector (iii), thereby generating a vector that expresses said protein of interest; and purifying said modified protein of interest by binding said protein of interest to said metal-modified matrix (iv).
 68. The process of claim 67, wherein said expression vector (iii) comprises a mammalian expression vector, a bacterial expression vector, an insect cell expression vector and a yeast expression vector.
 69. The process of claim 67, wherein said expression vector (iii) comprises a plasmid or viral vector.
 70. The process of claim 67, wherein said purifying step, the metal-modified matrix comprises a chromatographic column or a microtitre plate.
 71. A fusion protein comprising an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for a different antibody.
 72. A fusion protein comprising an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for a different antibody.
 73. The fusion protein of claim 71, wherein said peptide affinity group comprises S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
 73. The fusion protein of claim 62, wherein said metal binding peptide comprises oligohistidine or an oligopeptide comprising the sequence HGGHHG (SEQ ID NO: 1, SPHHG (SEQ ID NO: 2), SPHHGGSPHHG (SEQ ID NO: 3), HPHHG (SEQ ID NO: 4), HPHHGGHPHHG (SEQ ID NO: 5), SPHHGGHPHHG (SEQ ID NO: 6), HPHHGGSPHHG (SEQ ID NO: 7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10) or HGLGHGHEQQHGLGHGH (SEQ ID NO: 9).
 74. A process for isolating an analyte of interest, said process comprising the steps of: providing (i) a sample containing or suspected of containing said analyte of interest; (ii) a first antibody having an affinity for said analyte; (iii) a fusion antibody comprising: (a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for a different antibody; or (b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for a different antibody; and (iv) a matrix comprising a metal or a second member of said affinity peptide group. contacting said sample with said first antibody, thereby forming a first complex between said first antibody and any analyte present in said sample; contacting said first complex with said fusion antibody, thereby forming a second complex between said first complex and said fusion antibody; contacting said second complex with said matrix to bind said second complex to said matrix; removing unbound material from said matrix, and releasing said analyte of interest from said second complex, thereby isolating said analyte of interest.
 75. The process of claim 74, wherein said peptide affinity group comprises S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
 76. The process of claim 74, wherein said metal binding peptide comprises oligohistidine or an oligopeptide comprising the sequence HGGHHG (SEQ ID NO: 1), SPHHG (SEQ ID NO: 2), SPHHGGSPHHG (SEQ ID NO: 3), HPHHG (SEQ ID NO: 4), HPHHGGHPHHG (SEQ ID NO: 5), SPHHGGHPHHG (SEQ ID NO: 6), HPHHGGSPHHG (SEQ ID NO: 7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10) or HGLGHGHEQQHGLGHGH (SEQ ID NO: 9).
 77. A process for detecting or quantifying an analyte of interest, said process comprising the steps of: providing (i) a labeled sample containing or suspected of containing labeled analyte of interest; (ii) a first antibody having an affinity for said analyte; (iii) a fusion antibody comprising: (a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for a different antibody; or (b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for a different antibody; and (iv) a matrix comprising a metal or a second member of said affinity peptide group; contacting said sample with said first antibody, thereby forming a first complex between said first antibody and any analyte present in said sample; contacting said first complex with said fusion antibody, thereby forming a second complex between said first complex and said fusion antibody; contacting said second complex with said matrix to bind said second complex to said matrix; removing unbound material from said matrix; and detecting or quantifying said labeled analytes bound to said matrix by means of detecting or quantifying a signal from said labels.
 78. The process of claim 77, wherein said peptide affinity group comprises S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
 79. The process of claim 77, wherein said metal binding peptide comprises oligohistidine or an oligopeptide comprising the sequence HGGHHG (SEQ ID NO: 1), SPHHG (SEQ ID NO: 2), SPHHGGSPHHG (SEQ ID NO: 3), HPHHG (SEQ ID NO: 4), HPHHGGHPHHG (SEQ ID NO: 5), SPHHGGHPHHG (SEQ ID NO: 6), HPHHGGSPHHG (SEQ ID NO: 7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO: 10) or HGLGHGHEQQHGLGHGH (SEQ ID NO: 9).
 80. The process of claim 77, wherein said analyte of interest comprises a protein or polypeptide.
 81. The process of claim 77, further comprising the step of releasing said analyte of interest from said second complex prior to said detecting or quantifying step.
 82. The process of claim 77, wherein said labeled analytes are detected fluorescently, chemiluminescently, colorimetrically or enzymatically.
 83. A process for detecting or quantifying an analyte of interest, said process comprising the steps of: providing (i) a labeled sample containing or suspected of containing labeled analytes of interest; (ii) a first antibody having an affinity for said analyte; (iii) a fusion antibody comprising: (a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for a different antibody; or (b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of said antibody, wherein said affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein said antibody has an affinity for a different antibody; and (iv) a matrix comprising a metal or a second member of said affinity peptide group. forming a first complex among said matrix, said fusion antibody and said first antibody; contacting said first complex with said labeled sample, thereby forming a second complex between said first complex and any labeled analytes that may be present in said sample; removing unbound material from said matrix; and detecting or quantifying said labeled analytes bound to said matrix by means of detecting or quantifying a signal from said labels.
 84. The process of claim 83, wherein said matrix comprises an array of different first antibodies, thereby detecting or quantifying multiple analytes of interest.
 85. The process of claim 83, wherein said labeled analytes are detected fluorescently, chemiluminescently, colorimetrically or enzymatically. 